The principal objective of this grant is to explore the factors n enzyme design that allow discrimination between substrates. Two enzymes, aspartate aminotransferase (AATase) and tyrosine aminotransferase (TATase), both convert amino acids into 2- oxoacids. The Former is restrictive in substrate selectivity to the dicarboxylic amino acids, L-Asp and L-Glu, while the latter is permissive and accommodates, additionally, aromatic amino acids. Specificity is controlled by the side-chain of Arg292, which is rigid in AATase to keep out aromatic amino acids, but is mobile in TATase, and permits access of nearly all varieties of amino acids. We will use genetic engineering, in combination with kinetic techniques, to try to understand how the mobility of the side chain is restricted by the protein superstructure. Further genetic engineering experiments will be employed to reverse substrate charge specificity, and thus, convert AATase to an arginine transaminase. This will involve additional structure-based mutations of the ARG292 Asp mutation that we investigated nine years ago. Collaborative work with crystallographers in Switzerland is directed to structure elucidation of two new pyridoxal phosphate dependent enzymes, TATase discussed above, and ACC synthase, the key enzyme controlling ethylene production in plants. Ethylene is the plant ripening and senescence hormone. Additional goals are 1) to understand the details of the energetics of the ACC synthase mechanism by determining how the important intermediate on the reaction pathway partitions back to reactants or on to products; 2) to determine the transition state structure for the abstraction of the C alpha proton of S-adenosyl methionine in ACC synthase by Bronsted analysis; 3) to evaluate the significance of an unusual so- called, low barrier hydrogen bond in AATase by amino acid modification; 4) to develop a definitive technique to diagnose the existence of substrate channeling, whereby the product of the Nth enzyme in a metabolic pathway is delivered directly to the Nth+1 enzyme, for which it is a substrate, rather than being released first into solution.
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